Combination antiretroviral therapy (cART) has slowed the onset, but has not altered the prevalence of HIV-associated neurologic disease (HAND). In aging patient populations, the prevalence of HAND is increasing, despite the ability of cART to reduce viral load in the CNS to undetectable levels. Half of the population living with HIV may experience symptoms of HAND. Development of more CNS penetrant antiviral drugs may be counter-productive, since it has been shown that antiretrovirals with good CNS penetrance are associated with impaired neurocognitive function. This may be a result of direct or indirect neurotoxicity, possibly resulting from induced metabolic states that favor pro-inflammatory vascular disease. The hallmarks of HAND include: 1) a dysregulation of inflammatory cytokines and chemokines, 2) the recruitment of immune cells (monocytes) to the CNS, 3) the viral infection of glia leading to interruption of their normal function, and 4) extensive synaptodendritc damage. A host of inflammatory mediators are implicated in this cascade, but a limited subset of mediators has been identified as being up-regulated in the cerebrospinal fluid and postmortem brain tissues of HAND patients: TNF-??, the monocyte chemoattractant MCP-1, and mixed-lineage kinase 3 (MLK3), an important control point in MAPK kinase regulated inflammation pathways. Previously in collaboration with Dr. Harris Gelbard and Stephen Dewhurst at the University of Rochester Medical Center, we have identified URMC-099, a first generation compound that inhibits MLK3 and blocks up-regulation of MCP-1, TNF-? and other inflammatory mediators in HIV-1 Tat stimulated human macrophages. In vivo brain imaging experiments in mice exposed intracerebrally to HIV-1 Tat have shown that i.p. administration of URMC-099 prevents Tat-induced leukocyte infiltration and microglial activation, and reverses Tat-induced damage to synaptic architecture. Damage to synaptic architecture is the key pathological process of HAND in cognition and memory impairment. URMC-099 is a selectively non-selective kinase inhibitor and inhibits a large number of protein kinases, a property that may hinder future development. In our Phase 1 SBIR research we sought to identify second generation, much more specific MLK3 inhibitors, that would protect neurons in in vitro models of microglial activation by HIV-Tat. We have successfully discovered a series of potent and much more selective MLK3 inhibitors, exemplified by CLFB-1134, with excellent in vitro activity in neuron protection assays, excellent blood brain barrier penetration, and satisfactory in vitro safety profiles for CYP450s, hERG and genotoxicity. In Phase 2 research we will optimize these compounds for MLK3 potency, specificity, oral bioavailability and activity in mouse brain imaging models of HIV-Tat challenge. Compounds showing efficacy in animal models will be profiled in in vitro safety and genotoxicity screens and dose ranging toxicity studies for selection as development compounds for late stage preclinical testing.